Sequence-specific DNA-binding nuclear proteins play a fundamental role in regulating gene expression. It would thus be valuable to develop a method permitting genomic scale differential cloning of such molecules. Current methods such as differential screening and construction of subtracted cDNA libraries fail to discriminate which of the differentially expressed genes encode nuclear DNA-binding proteins. To identify such proteins, the investigators propose using a functional genomics approach: The first step referred to as """"""""nuclear trapping"""""""" consists of isolating nuclear proteins encoded by a random primed subtracted cDNA library flanked with a C-terminal tag that encodes six histidine (His) residues. Transfection of this library into cell-lines will permit transcription and translation of cDNAs that have complete 5' ends. Nuclear proteins encoded by some of these cDNAs will localize to the nucleus where they can be purified from a nuclear extract with nickel-agarose beads through their His-tagged C-terminus. The second step couples multiple rounds of specific affinity enrichment and polymerase chain reaction (PCR) selection to identify DNA sequences that are recognized by the isolated pool of His-tagged nuclear proteins. Finally, the selected pool of target sequences can be used to clone cDNAs that encode proteins that bind to these sites. The investigators will refer to PCR affinity selection expression cloning using the acronym PASEC. The goal of this proposal is to perform a pilot project that will assess the feasibility of developing technology permitting genomic scale differential cloning of cDNAs encoding nuclear proteins that bind double stranded (ds) DNA in a sequence-specific manner. Though feasibility studies will be performed using as a marker a Drosophila melanogaster ovarian protein whose nuclear localization and DNA-binding specificity are well characterized, the method is versatile and can be adapted for use in any eukaryotic system.
The specific aims i nclude: 1. Assess the feasibility of nuclear trapping by substituting a single cDNA that encodes the ovarian transcription factor CF2-II protein (instead of a differential subtracted cDNA library) during the transfection and His-tag purification phase of the procedure. 2. Assess the feasibility of the PASEC procedure using the His-tagged CF2-II protein purified from the nuclei of transfected cell lines. 3. Perform 20 rounds of specific affinity enrichment and PCR amplification to obtain pools of DNAs recognized by site-specific DNA binding proteins contained within Drosophila ovarian nuclear extracts. Since CF2-II is expressed in the nuclei of ovarian follicle cells, the investigators will determine whether any of the amplified pools of DNA contain known CF2-binding sites.
| Bai, C; Tolias, P P (2000) Genetic analysis of a La homolog in Drosophila melanogaster. Nucleic Acids Res 28:1078-84 |
